The X-ray crystal structure and biochemical analysis of a native basic pathogenesis-related-1 protein from Mucuna sempervirens (Fabaceae) floral nectar

Abstract Background and Aims Pathogenesis-Related 1 (PR-1) proteins are small, secreted proteins that typically accumulate upon pathogen attack and serve as hallmarks of plant immune activation. Despite their widespread use as defense markers, their biochemical properties and native structural features remain largely unresolved. This study aimed to isolate, characterize, and determine the crystal structure of a native PR-1 protein (MsPR-1b) that predominates in the floral nectar of Mucuna sempervirens, a woody leguminous vine, to provide insight into the molecular diversity and possible functional specialization of PR-1 proteins. Methods MsPR-1b was purified from raw nectar using ion-exchange and size-exclusion chromatography. The purified protein was analyzed for molecular mass and glycosylation status, and its three-dimensional structure was determined by X-ray crystallography at 2.0 Å resolution. The full-length cDNA sequence was obtained through RACE PCR, and tissue-specific expression was examined by quantitative PCR. Lipid-binding ability and antimicrobial activity were assessed by fluorescence spectroscopy and the disk diffusion method, respectively. Key Results The MsPR-1b gene encodes a 164-amino-acid precursor containing an N-terminal signal peptide. Expression was predominantly detected in the nectary. The mature MsPR-1b is a small, basic, secreted protein (15,134 Da, pI 9.4) that lacks glycosylation. The crystal structure, representing the first native PR-1 protein purified from its natural source revealed a homodimer. Each monomer adopts the conserved α–β–α sandwich fold characteristic of the CAP superfamily and contains two intrachain disulfide bonds. MsPR-1b displays no detectable lipid-binding capacity or direct antimicrobial activity. Conclusions MsPR-1b provides the first structural view of a native plant PR-1 protein and indicates that nectar-secreted PR-1s may perform specialized, non-defensive functions distinct from canonical pathogen-related roles.